Human Artificial Chromosomes (HACs) assembled from alphoid DNA arrays represent novel vectors that have a great potential for the study assembly and maintenance of human kinetochore as well as for gene therapy, screening of anticancer drugs and biotechnology. We previously constructed a synthetic HAC (tetO-HAC) allowing tethering of its kinetochore by different chromatin modifies fused with the tet-repressor protein. This HAC was successfully used to clarify role of different types of chromatin in functioning of the kinetochore. During past year, the same approach based on tethering of the tetO-HAC kinetochore by different fusions revealed that mitotic transcription plus histone modifications including H3K9ac constitute the 'epigenetic landscape' allowing CENP-A assembly and centrochromatin maintenance. H3K4me2 is required for the transcription and H3K9ac may form a barrier to prevent heterochromatin spreading and kinetochore inactivation at human centromeres. In another study, we demonstrated that human centromere resists silencing mediated by H3K27me3/K9me3. In separate experiments, a platform with multi-integrase recombination sites has been inserted into tetO-HAC and has been successfully used for a gene assembly in the HAC. Work is in progress to assemble synthetic nucleolar organizer region (NOR) in the HAC using TAR-isolated human rDNA units. Such a HAC module that is transferable into any type of human cells via micro-cell mediated chromosome transfer (MMCT) will be used to investigate the requirements for nucleolar location of rDNA repeats and effect of copy number of rDNA units on cell proliferation and stress response. We have also applied our tetO-HAC for measuring chromosome instability (CIN) in human cells. Whole-chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a characteristic feature of most types of cancer, thus distinguishing them from their normal counterparts. Although CIN is generally considered a driver of tumor growth, a threshold level exists whereby further increase in CIN frequency becomes a barrier against tumor growth and therefore can be exploited therapeutically. However, drugs known to increase CIN beyond this therapeutic threshold are currently few in number. In our previous work, we have developed a new quantitative assay for measuring CIN based on the use of a non-essential HAC carrying a constitutively expressed EGFP transgene. Thus, cells that inherit the HAC display green fluorescence, while cells lacking the HAC do not. This allows measurement of HAC loss rate in response to drug treatment by routine flow cytometry. We used this assay to rank more than 100 anticancer drugs on their effect on HAC loss. The strongest effect was observed for, taxol (microtubule-stabilizing agent), LMP744 (inhibitor of topoisomerase TOP1, developed in our branch). We also demonstrated the utility of the assay to detect increase of CIN after siRNA depletion of known genes controlling chromosome transmission. In our recent work, we modified EGFP-HAC and converted the original assay into high-throughput CIN screen of chemical libraries and siRNA libraries of human genes. Analysis of siRNAs targeting each of 720 human protein kinase genes revealed 27 CIN genes with no information on their role in chromosome transmission. Each of these new CIN genes may be considered as a new target for cancer therapy.